In lymph node negative breast cancer, a strong association has been shown between S-phase and prognosis. In addition, demonstration of an aneuploid population has diagnostic implications. We studied the effect of doublet discrimination on S-phase fraction estimates and detection of tetraploid populations in fifty breast carcinomas, peripheral blood lymphocytes and cell lines. Doublet estimates obtained using computer modeling as well as electronic detection techniques based upon pulse shape analysis of fluorescent height vs area or fluorescent width vs area were compared. In biologically homogeneous (eg cell lines) specimens, all techniques yielded good results. However, in biologically complex specimens doublet estimates varied greatly (up to five fold difference) depending upon the method used. This variation in doublet subtraction affected estimates of proliferating cells. Light chain restriction (detection of monoclonal B-cells) is synonymous with B-cell neoplasia in most clinical situations. We therefore studied flow cytometric methods for light chain detection in 375 patients and 181 normal controls. We detected monoclonality in 92 of the 375 patient specimens and in no normal controls (results correlated with clinical outcome). 79 out of 375 (23%) specimens were grossly monoclonal and detected by standard methods. Of these 79 cases, different sets of anti-light chain reagents had different sensitivities with one detecting all monoclonal populations but sensitivities of 66% and 78% obtained with two other sets. In 24 cases negative for monoclonality by standard methods but in which the patient had a previous history of lymphoma, a clonal search was performed based upon antigen density and cell size. Monoclonality was detected in 13 of these 24 cases (54%) demonstrating the superior sensitivity of this method. We have established flow cytometric methods to quantitate expression of c-myc, bcl-2 and p53 protein expression. We will compare these methods to RT-PCR methods on sorted and unsorted patient specimens.